ORIGINAL ARTICLE

Stabilization of Gas Bubbles Released from Water-SolubleCarbohydrates Using Amphiphilic Compounds: Preparationof Formulations and Acoustic Monitoring of Bubble Lifetime

Lars Hoff • Per A. Foss • Knut Dyrstad •

Jo Klaveness • Pal Rongved

Received: 20 July 2010 / Accepted: 20 January 2011 / Published online: 13 February 2011

� The Author(s) 2011. This article is published with open access at Springerlink.com

Abstract The ultrasound contrast agents Echovist� and

Levovist� (Bayer AG, Schering AG, Germany) are based

on the release of gas bubbles from milled a-D-galactose. In

diagnostic ultrasound, for this class of contrast agents, there

is a need for prolonged contrast duration. To investigate if

new carbohydrate compositions could prolong the lifetime

of the gas bubbles, a-D-galactose was mixed with other

carbohydrates or amphiphiles with varying log P. Acoustic

attenuation vs. time (390 s) area under the curve (A390) and

bubble half-time (t�) were used as measures of prolonged

lifetime of gas bubbles. The products, to which 0.1% of a

lipophilic carboxylic acid (5b-cholanic acid, behenic acid,

and melissic acid) has been added, showed more than 5, 7

and 11 times enhancement of A390, respectively, compared

with the reference compound 2 (RC2) corresponding to the

commercial product Levovist�. The half-time t � of the

same compounds was prolonged more than 6 times com-

pared with RC2. A partial least square (PLS) statistical

analysis confirmed that, for additives, high log P carboxylic

acids lead to the highest A390. The present results bear a

promise of products with a more persistent in vivo ultra-

sound contrast effect than the commercially available

agents.

Keywords Gas bubbles � Ultrasound � Contrast agents �Carbohydrates � Gas-release � Amphiphiles � Colloid �Fatty acids � Acoustics

Introduction

Gas bubbles in the micrometer range are ideal contrast

agents for ultrasound imaging because of their high com-

pressibility and low density, giving excellent reflectance of

ultrasound waves (acoustic backscatter) [1]. Ultrasound

contrast signals from free gas bubbles in vivo in the human

heart were first observed in 1968 after direct intracardiac

injection [2]. Short bubble lifetime urged development of

more stable agents giving more persistent contrast on the

arterial side after intravenous injection and passage through

the lung capillaries. The ultimate goal is to generate contrast

in the heart muscle (myocardium). Suspensions of solid

particles, emulsified liquid droplets, free and encapsulated

gases or liquids [3] have been proposed as ultrasound

contrast agents (USCA). Efficient USCA may comprise

micron-sized bubbles stabilized by a thin, potentially bio-

degradable flexible membrane consisting of polymers [4] or

phospholipids [5]. However, carbohydrate-based gas-

releasing systems, generating free gas bubbles after intra-

venous injection of a particle suspension, eliminate the

challenge of developing a stabilized bubble suspension as a

J. Klaveness � P. Rongved

School of Pharmacy, University of Oslo,

P.O. Box 1155, 0316 Blindern, Oslo, Norway

K. Dyrstad

GE Healthcare ASA, P.O. Box 4220,

0401 Nydalen, Oslo, Norway

P. A. Foss

Hunt Biosciences, Halsanvegen 24,

7600 Levanger, Norway

L. Hoff

Høgskolen i Vestfold, Postboks 2243,

3103 Tønsberg, Norway

P. Rongved (&)

Department of Pharmaceutical Chemistry,

School of Pharmacy, University of Oslo,

P.O. Box 1068, 0316 Blindern, Oslo, Norway

e-mail: pal.rongved@farmasi.uio.no

123

J Surfact Deterg (2011) 14:585–593

DOI 10.1007/s11743-011-1250-y

drug product, with demands of long term shelf stability. In

the commercially available agent SHU 454 (Echovist�,

Schering AG, Berlin), milled, particulate water-soluble

a-D-galactose acts as a precursor for gas bubbles, and is in

clinical use in enhanced hystero-sonography [6]. The gas-

releasing powder is prepared using ball-milling of com-

mercial a-D-galactose, giving clusters of particles with a

mean size of about 40 lm, with air-filled voids between

them [7]. Upon dissolution in water, the clusters release the

air trapped in the clusters and gas bubbles are generated.

The a-D-galactose crystals dissolve in the water phase while

acting as short-living nucleation sites for the gas bubbles

[7]. Lack of myocardial contrast enhancement (MCE) with

SHU 454 in humans led to the improved agent SHU 508

(Levovist�, Schering AG, Berlin), wherein a-D-galactose is

milled with 0.1% palmitic acid [8]. However, in spite of

more persisting contrast on the arterial side of the heart with

SHU 508 [9], it is not in clinical use to study early reduced

blood flow in the myocardium using MCE. Since pre-

formed, lyophilized phospholipid-stabilized gas bubbles on

the market have been a success [10], the simplicity of the

gas-releasing carbohydrate-based products in the form of a

dry, particulate, water-soluble precursor of gas bubbles

encouraging further research. In the present research, our

first aim was to investigate if lifetime of the populations of

gas bubbles released could be prolonged, by investigating a

broad range of amphiphilic compounds. Our second aim

was to investigate if acoustic attenuation vs. time (390 s)

area under the curve could be used as a suitable screening

tool in this research.

Experimental Section

Materials

Chemicals

Starch was purchased from Reppal PSM 70, Reppe Glykos

(Sweden). a-D-Xylose was purchased from BDH (Basel,

Switzerland). 1,2-dipalmitoyl-sn-glycero-3-phosphati-

dylcholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phos-

phatidylethanolamine (DOPE) were purchased from Avanti

Polar Lipids, USA. Human serum albumin (HSA), sodium

dodecyl sulfate (Sodium lauryl sulfate, Na-LS), 2-sul-

fobutanedioic acid 1,4-bis(2-ethylhexyl) ester sodium

salt (Na-docusate), Pluronic� F68 (Mw 8.4 kDa) a very

hydrophilic polyoxyethylene polyoxypropylene triblock

copolymer (abbreviated HTBCP), sorbitan mono-oleate

isomer mixture (Span 80) abbreviated SMO, sorbitan tri-

oleate isomer mixture Span 85) abbreviated STO, a-lino-

lenic acid ((Z,Z,Z)-9,12,15-octadecatrienoic acid) and other

fatty acids were purchased from Sigma Chemical Company

(St. Louis, MO). Tricosa-10,12-diynoic acid was purchased

from ABCR GmbH & Co. (Karlsruhe, Germany). Hexa-

decanedioic acid, a-cyclodextrin, dextran (Mw 20 kD),

maltodextrin, glycogen, a-D-galactose and all other chem-

icals were purchased from Fluka Chemie AG (Buchs,

Switzerland) or E. Merck AG (Darmstadt, Germany).

Scheme 1 gives the structures of the used compounds

except for the oligo and polysaccharides. Deionized water

was used throughout the experiments. Chemical structures

and names of the compounds used in the present work are

given in Scheme 1.

Visual Inspection of the Products After Suspension

in the Carrier Liquid

Screening of the rough ability of the products to release gas

microbubbles was performed using conventional light

microscopy.

Area Under the Curve of Acoustic Attenuation

and Bubble Half-Time

A carrier liquid for determining the acoustic attenuation of

the products consisted of 10 mL of propylene glycol mixed

with 90 mL of 5% dextrose in water. Each product (1.0 g)

was dispersed in the carrier liquid (3.0 mL) and gently

handshaken for 15 s. The resulting mixture was added to

5% human serum albumin in phosphate buffer (52 mL).

The mixture was placed in a measurement cell with a

5 MHz broadband transducer mounted on one side wall,

and an ultrasound-reflecting plate on the opposite side of

the cell. A pulse-reflection technique measuring the

acoustic transmission through the product dispersions was

used to calculate the acoustic attenuation taken as the

inverse value of the measured acoustic transmission. The

acoustic attenuation (dB/cm) was plotted as a function of

time for 390 s. Temperature in the measurement cell was

37 ± 1 �C with constant circulation of the liquid. The

results were normalized with regards to measurements of a

reference consisting of 55 mL of 5% human serum albu-

min buffer solution. The calculated integral of the area A

under the curve of acoustic attenuation vs. time (390 s),

was denoted A390. This parameter expressed the total

amount of gas phase in the dispersions up to and including

390 s. Average bubble lifetime was characterized by the

half-time (t�) of the acoustic attenuation up to 390 s. Both

A390 and t� were necessary to decide if persistence of

ultrasound contrast effect in vitro was improved for the

various formulations compared with the reference stan-

dards (defined below).

586 J Surfact Deterg (2011) 14:585–593

123

Particle Size Distribution

The particle size distribution of selected products was

analyzed using a Coulter Counter LS 100 or a Malvern

Mastersizer light-scattering apparatus by suspending

2 g of the product using a vibrating screen before

measuring.

Monitoring of Amphiphiles

The surfactants, phospholipids and fatty acids milled with the

carbohydrates were monitored by analyzing the product

(1.0 g) in carrier liquid (3 mL) using thin layer chromatog-

raphy (TLC) and detecting spots using Nile blue fluorescence

and copper complexation as described in the literature [11].

Scheme 1 Chemical structures and names of the compounds used. The structures of starch, a-cyclodextrin, dextran Mw 20 kD, maltodextrin and

glycogen are omitted for clarity

J Surfact Deterg (2011) 14:585–593 587

123

log P Values

The concept of hydrophilic-lipophilic balance (HLB) is

based on calculations of the ratio of hydrophilic and lipo-

philic groups in the molecules [12]. However, HLB values

approach zero for many of the compounds used in the

present study with low water solubility. Therefore calcu-

lated log P was found to be a more useful parameter char-

acterizing the amphiphiles used herein. The log P values of

the amphiphilic compounds were calculated using the

chemist drawing and calculation software ChemBioDraw,

version 11.0.1, CambridgeSoft Europe Office, 1 Signet

Court, Swanns Road, Cambridge CB5 8LA UK.

Data Analysis

The parameters defined below were used (abbreviations in

parentheses) in a partial least squares analysis (PLS) model

with A390 as a response parameter: % (w/w) amphiphilic

material added (% amph); calculated log P of the amphi-

philes (log P); ionic character in sodium salt form (ionicity:

0 or 1); mean particle size in freshly prepared suspensions

(psize); % (w/w) a-D-galactose (%Gal); molecular weight

of the amphiphilic additive (LipidMw); degree of unsatu-

ration in the additives as number of double bond equiva-

lents (Unsat: 1–4); number of rings in the chemical

structure of the amphiphile (rings); presence of carboxylic

acid groups (Carboxyl: 0 or 1). The PLS analysis was

performed using the computer program Unscrambler, ver-

sion 7.01 from CAMO Software AS, Oslo, Norway.

Preparation of Products

All concentrations are given as % w/w in the procedures.

Two literature procedures for preparation of carbohydrate-

based gas-releasing ultrasound contrast agents [7, 13] were

modified to establish the three general preparation proce-

dures below. Ball-milling was performed with a Retsch

centrifugal ball-mill in a stainless steel ball-mill having a

50 mL grinding cup and 3 9 20 mm balls for 10 min. All

products appeared as white powders after milling.

General Procedure I: Mixtures of Carbohydrates Without

Amphiphiles Added, Products 1–4

Products (1) and (2) consisted of commercial qualities of

a-D-galactose (1) and a-D-xylose, respectively, that were

ball-milled. The a-D-galactose/Starch Mixtures (3a-c) were

prepared by mixing a-D-galactose (3.2, 2.0 and 0.8 g) with

starch (0.8, 2.0, and 3.2 g, respectively) followed by ball-

milling. The a-D-galactose/Dextran (Mw 20 kD) mixtures

(4a-b) were prepared by combining 25.8% solutions of

a-D-galactose in water (29.2 and 19.4 g) with 25.7%

solutions of Dextran in water (9.7 and 19.4 g, respectively).

The combined solutions were evaporated to dryness while

stirring under reduced pressure (10 Torr, 40 �C) and dried

in a desiccator overnight. The products were then ball-

milled. Experimental data and results for products 1–4 are

given in Table 1.

General Procedure II: a-D-galactose-Based Products

with Surfactants (5–11) and Lipophilic Acids (12–19)

For each of the products 5–19, the amphiphilic additives

were each dissolved in 96% ethanol or water at 50–78 �C.

The resulting solutions were filtered and then added to 1

(24.2 g) under stirring. The resulting mixtures were evap-

orated to dryness under reduced pressure (10 Torr, 40 �C)

and the resulting solid products were dried in a desiccator

overnight. The residues were ball-milled to give the final

products. Experimental data and results for products 5–19

are given in Tables 2 and 3.

General Procedure III: Palmitic Acid Added to a Mixture

of a-D-galactose (1) and Starch (20) and Carbohydrates

Other than a-D-galactose (21–24)

For 20, a-D-galactose (1, 12.1 g) was heated to 60 �C and

mixed with a 14.3% w/w starch solution in water (35.0 g)

before addition of the fatty acid solution. For products 21

and 22, a-D-galactose (1) was replaced by 41.3% w/w

solutions (24.2 g) of a-D-xylose and maltodextrin, respec-

tively. For 23 and 24, a-D-galactose (1) was replaced by

22.5% w/w solutions (22.2 g) of glycogen and a-cyclo-

dextrin, respectively. The resulting mixtures were evapo-

rated to dryness under reduced pressure (10 Torr, 40 �C).

The resulting solid products were dried in a desiccator

overnight and the residues were ball-milled to give the

products. Experimental data and results for products 20–24

are given in Table 4.

Table 1 A390 for products 1–4 without addition of amphiphiles

a-D-galactose ? carbohydrate 2 (C2) Prod. % C2 A390 t � (s)

a-D-galactose (reference 1, RC1) 1 0 5 16

a-D-xylose 2 0 &0 0

a-D-galactose ? Starch 3a 20 164 30

3b 50 161 35

3c 80 70 38

a-D-galactose ? Dextran 4a 25 23 54

4b 50 13 48

Milled a-D-galactose (1) is reference product 1 (RC1) and corre-

sponds to the commercial product Echovist�. Carbohydrate 1 is

milled a-D-galactose or a-D-xylose. The added carbohydrate is

denoted C2

588 J Surfact Deterg (2011) 14:585–593

123

Results and Discussion

Results

The particle size distributions of the solid products 1–24

were in the range of 1–20 lm (Coulter Counter, data not

given). In example, a typical accumulated particle volume

distribution of 18b showed that 90% was \4.4 lm and

10% was \0.5 lm, the mean diameter was 2.2 lm and the

median diameter was 2.1 lm. Based on visual microscopy

observation, the solid particles tended to release air bubble

dispersions in the size range of 1–15 lm, with some larger

bubbles.

For product comparison, two reference compounds were

needed: according to the literature and product descriptions

[7, 13, 14], milled a-D-galactose (1) corresponds to the

commercial product Echovist� and was chosen as refer-

ence product 1 (denoted RC1). According to the literature

[8] and public information, the product consisting of a-D-

galactose milled with 0.1% palmitic acid (13a) corresponds

to the commercial product Levovist�, and was chosen as

reference product 2 (denoted RC2). The results of the

acoustic characterization expressed as A390 are given in

Tables 1, 2, 3 and 4. The results of the measurement of

A390 after mixing with the carrier liquid are given in

Figs. 1, 2, 3, 4 and 5.

Table 2 A390 for products 5–11comprising a-D-galactose,

milled with amphiphilic

compounds lacking a carboxylic

acid functionality

For each product, abbreviation

or trivial name of the

amphiphile (A) is followed by

formula, molecular weights and

calculated log P in parentheses.

In the last three columns,

amounts of A, A390 values and

half-times are given. The

chemical names of the

amphiphiles are given under

Materials and Methods

Amphiphile A with trivial name (formula, Mw, log P)

milled with a-D-galactose

Prod. % A A390 t� (s)

DPPC (C40H81NO8P, 736.1, 9.8) 5a 0.1 366 53

5b 1.0 3,712 [500

Na-LS (C12H25NaO4S, 266.4 (anion), 1.8) 6a 0.1 22 23

6b 1.0 67 66

SMO (C24H46O7, 446.6, 5.0) 7a 0.1 &0 0

7b 1.0 34 26

STO (C60H110O9, 975.5, 21.2) 8a 0.1 6 34

8b 1.0 28 72

HTBCP (Average Mw 8,400, log P n.a.) 9a 0.1 58 21

9b 1.0 74 23

Na-Docusate (C20H37NaO7S, 422.6 (anion), 6.1) 10a 0.1 55 23

10b 1.0 3 0

DOPE (C44H86NO8P, 744.1, 13.6) 11a 0.1 343 [500

11b 1.0 552 72

Table 3 A390 values of

products 12–19 comprising a-D-

galactose,

milled with amphiphilic

compounds possessing a

carboxylic acid functionality

Milled a-D-galactose milled

with 0.2% palmitic acid (13b) is

reference product 2 (RC2) and

corresponds to the commercial

product Levovist�. For each

product, abbreviation or trivial

name of the carboxylic acid C is

followed by the formula,

molecular weight and calculated

log P in parentheses. In the last

three columns, amounts of A,

A390 values and half-times are

given. The chemical names of

the amphiphiles are given under

‘‘Materials and methods’’

Carboxylic acid C, trivial name (formula, Mw, log P)

milled with a-D-galactose

Prod. % C A390 t� (s)

Capric acid (C10H22O2, 172.3, 4.0) 12a 0.1 &0 0

12b 1.0 &0 0

Palmitic acid (C16H34O2, 256.4, 7.0) reference 2

(RC2) = 13a (0.1% palmitic acid)

13a 0.1 357 31

13b 0.2 871 35

13c 1.0 1,061 107

Hexadecane-dioic acid (C16H34O4, 286.4, 5.1) 14 1.0 474 40

Linolenic Acid (C18H30O2, 278.4, 7.3) 15 1.0 401 79

Behenic acid (C22H44O2, 340.6, 9.9) 16a 0.1 2,745 [500

16b 1.0 4,653 [500

10,12-tricosa-diynoic acid (C23H38O2, 346.6, 9.9) 17 0.2 884 51

Melissic acid (C30H60 O2, 452.8, 13.8) 18a 0.01 322 150

18b 0.1 4,095 [500

18c 1.0 4,564 [500

5b-cholanic acid (C24H40O2, 360.6, 7.7) 19a 0.01 162 47

19b 0.1 1,962 203

19c 1.0 4,048 [500

J Surfact Deterg (2011) 14:585–593 589

123

Figures 6 and 7 show the statistical analysis of the

results, using A390 as a response parameter. A high log P,

the presence of a-D-galactose and that of a carboxylate

group in the amphiphilic molecule correlates positively

with A390. In addition, the PLS analysis reveals some

interaction effects between parameters. There is a strong

interaction between log P and the presence of a carboxylate

group; an interaction between log P and the presence of

a-D-galactose is also evident from the analysis.

Discussion

As shown in Figs. 1 and 2, the use of other carbohydrates

than a-D-galactose does not improve performance (A390 and

t�) as compared with the a-D-galactose-based reference

products RC1 and RC2. The observed significant increase

in both A390 and t� when a-D-galactose is mixed with 0.1%

of b-cholanic acid (19b), behenic acid (16a), and melissic

acid (18b) compared with RC2 (Fig. 5), correlates well

with the log P values for the acids. The products 16a and

18b show half-time t� [ 500 s compared with t� = 31 s

for RC2 (Table 3). For instance, log P for 5b-cholanic,

behenic and melissic acids are calculated to be 7.7, 9.9 and

13.8, respectively. The A390 values for the corresponding

products 19b, 16a and 18b are 1,962, 2,745 and 4,095,

respectively. When the amount of these fatty acids is

increased to 1% (products 16b, 18c and 19c, Table 3,

Fig. 4) the correlation of A390 and t� with log P is even

more pronounced.

An interesting comparison is the results for the products

13c, 14 and 15, with 1% (w/w) palmitic acid, hexadec-

anedioic acid and linolenic acid, respectively (Table 3).

Both A390 and t� are more than 100% higher for 13c

Table 4 A390 values for products 20–24 different carbohydrates

milled with 0.2% palmitic acid

Carbohydrate milled with

palmitic acid

Product A390 t� (s)

a-D-galactose ? Starch 20 142 152

a-D-Xylose 21 706 245

Maltodextrin 22 254 225

Glycogen 23 358 [500

a-Cyclodextrin 24 94 47

Fig. 1 Acoustic attenuation (dB/cm) vs. time (390 s) of carbohydrate

mixtures without amphiphiles added, compared with the reference

standard 1 (RC1), milled a-D-galactose (1). When no amphiphiles are

present, mixing of a-D-galactose with polymeric carbohydrates like

starch or Dextran with Mw 20 kD only slightly increases A390

compared with RC1

Fig. 2 Acoustic attenuation (dB/cm) vs. time (390 s) of different

carbohydrates with 0.2% palmitic acid compared with the reference

standard 2 (RC2), milled a-D-galactose with 0.2% palmitic acid

(13b). Adding palmitic acid to different carbohydrates shows that

A390 of RC2 is superior for the first 100 s compared with the products

containing maltodextrin, glycogen and a-cyclodextrin with the same

amount of palmitic acid

Fig. 3 Acoustic attenuation (dB/cm) vs. time (390 s) of a-D-galact-

ose with 1% of different amphiphiles, surfactants and linolenic acid

compared with a-D-galactose containing 1% palmitic acid. The

intensity and duration of the acoustic attenuation of all products

containing 1% amphiphiles with various log P values, but lacking

carboxylic acid functionality, is significantly lower than for RC2

590 J Surfact Deterg (2011) 14:585–593

123

compared with 14. The dicarboxylic acid 14 will need to

bend its lipophilic chain at the gas–water interface,

reducing the lipophilic interaction. For linolenic acid (15),

a highly unsaturated carboxylic acid with a log P compa-

rable to palmitic acid, A390 is at the level of 14 and with a

half life of only 79 compared with 107 for 13c (Table 3 and

Fig 3). This may be due du the high number of cis double

bonds in linolenic acid, giving a non-linear conformation of

the lipophilic chain, reducing the hydrophobic interactions

between the hydrocarbon chains [15].

The PLS analysis illustrated by the regression coeffi-

cient plot (Fig. 6) provides further support for the trends

observed in the A390 plots. An absolute high and stable

regression coefficient for many principal components

indicates a robust effect of the actual descriptor (Fig. 6).

The strongest positive correlation is shown between am-

phiphiles possessing a carboxylic acid functionality and

high log P added to a-D-galactose. This points at fatty acids

with a number of carbon atoms [20, in good compliance

with the experimental data. These data are further

strengthened by the experimental results for amphiphiles

with high log P but lacking the carboxyl functionality; all

have low A390 (e.g. the phospholipid-based product 11).

Mw alone has negligible effect on A390 as shown by its

regression coefficient (Fig. 6). The good correlation

between predicted and observed A390 values (Fig. 7) shows

that a suitable model explaining the main effects on A390

has been established. The reliability of the PLS estimated

trends was also tested by regressing various logarithmic

transformations of the response having a more uniform

variation than the original A390, and unimportant regression

coefficients were successively removed. This process gave

similar regression coefficients patterns as shown in Fig. 6

regarding the most important parameters affecting A390 as

well as t�.

Ultrasonic waves are heavily attenuated at gas–water

interfaces [1]. It has earlier been reported [16] that a linear

correlation between ultrasonic attenuation and interfacial

area in a gas–liquid bubble column could be used to esti-

mate the total interfacial area in the system. The same

acoustic phenomenon was used in the present study to

establish a method to monitor the amount and lifetime of

bubble populations generated by milled carbohydrates.

The amphiphiles in the present study with the highest

log P are practically insoluble in water [17]. Thus, when

the ethanolic solutions of the amphiphiles are added to the

aqueous carbohydrate solutions (products 5–19), colloidal

suspensions may form. It was shown in a study of fatty acid

particle formation in water containing a surfactant [18] that

the particle size of such suspensions depended on the chain

length of the fatty acid. Gas bubbles have an affinity for

lipophilic particulate surfaces providing stabilization of the

gas phase [19–22], providing one possible mechanism for

the observed bubble stabilizing effect during the dissolu-

tion of the carbohydrate-amphiphile admixtures. However,

a more plausible mechanism is the formation of fatty acid

Langmuir films at the gas–water interface, systems exten-

sively described in the literature [17, 23–26]. If the

amphiphilic compounds are spread on the gas–water

interface, reduction of surface tension would result, as

shown by isotherm studies in the cited literature. Reduced

surface tension would increase bubble stability, explaining

the observations in the present study. It is also well known

from reports of investigation of aerosol systems that fatty

acids with high log P tend to be organized at the gas–liquid

as Langmuir films [17]. In that study, it was shown that

chain length and the carboxylic acid functionality were

important for the interfacial lifetime of the Langmuir films.

One question arising is how the fatty acids with the highest

log P and very limited solubility in water could achieve a

Fig. 4 Acoustic attenuation (dB/cm) vs. time (390 s) of a-D-galact-

ose with 1% of different amphiphiles, higher fatty acids and DPPC

Fig. 5 Acoustic attenuation (dB/cm) vs. time (390 s) of a-D-galactose

with 0.2% of different amphiphiles. When a-D-galactose is milled

with 0.1% of b-cholanic acid (19b), behenic acid (16a), and melissic

acid (18b), a significant increase in A390 compared with RC2 is

observed

J Surfact Deterg (2011) 14:585–593 591

123

dissolved state to form layers at the gas–water interface in

the carbohydrate formulations. The ultrasonic influence in

the present experiments may aid the dissolution process, as

described in literature for systems comprising substances

with low water-solubility [27].

Conclusion

In conclusion, the response parameters A390, expressing the

amount of gas-phase present during 390 s, and the bubble

half-time t� was shown to be useful main parameters in a

screening method for increased persistence of the ultrasound

contrast effect in vitro. Using these parameters, it was shown

that the persistence and amount of released gas bubbles in

formulations used in the commercial carbohydrate-based,

gas-releasing available and improved ultrasound contrast

agents (Echovist� and Levovist� respectively) could be

improved more than 10 times using fatty acids with a higher

log P relative to palmitic acid. Saturated fatty acids with

chain length higher than 20 carbon atoms are commercially

available, are found in many nutritional products and should

find convenient use in pharmaceutical products. Specifi-

cally, the increases in A390 and t� shown for product 19b-c,

and especially products 16a-b and 18b-c containing

5b-cholanic, behenic and melissic acid, respectively, are

results that encourage further research and in vivo studies.

Studies are ongoing to investigate the in vivo properties of

the most promising test substances.

Acknowledgments The authors wish to thank Nycomed Amersham

ASA for help and support of the present work.

-0.6

-0.3

0

0.3

0.6

log p

Ionicity

% G

al

Mw

Unsat

Rings

Carboxyl

log *Ioni

log *% G

a

log *Mw

log *Unsa

log *Ring

log *Carb

Ioni*% G

a

Ioni*Mw

Ioni*Unsa

Ioni*Ring

Ioni*Carb

% G

a*Mw

% G

a*Unsa

% G

a*Ring

% G

a*Carb

Mw

*Unsa

Mw

*Ring

Mw

*Carb

Unsa*R

ing

Unsa*C

arb

Ring*C

arb

Result, (Y-var, PC): (A390,3) (A390,4) (A390,5)

X-variables

Regression CoefficientsFig. 6 Results of the PLS

analysis including interaction

effects (regression coefficients)

between parameters derived

from principal components 3, 4

and 5 with A390 as response

parameter

-1000

0

1000

2000

3000

4000

0 1000 2000 3000 4000 5000 Result, (Y-var, PC): (A390,5)

5a6b7b8b10b

11a

12a

13a 13b13b

14

16a

17

18b

19b

20

21222324

Measured Y

Predicted YFig. 7 Correlation plot of the

observed vs. predicted in vitro

contrast effect expressed as A390

values using the present PLS

model

592 J Surfact Deterg (2011) 14:585–593

123

Open Access This article is distributed under the terms of the

Creative Commons Attribution Noncommercial License which per-

mits any noncommercial use, distribution, and reproduction in any

medium, provided the original author(s) and source are credited.

References

1. Goldberg BB (ed) (1997) Ultrasound contrast agents. Dunitz M

Pub, London

2. Gramiak R, Shah PM (1968) Echocardiography of the aortic root.

Invest Radiol 3:356–366

3. Klibanov AL (2002) Ultrasound contrast agents: development of

the field and current status. Top Curr Chem, 222(Contrast Agents

II):73–106

4. Eisenbrey JR, Burstein OM, Wheatley MA (2008) Effect of

molecular weight and end capping on poly(lactic-co-glycolic

acid) ultrasound contrast agents. Polym Eng Sci 48:1785–1792

5. Edey AJ et al (2008) Ultrasound imaging of liver metastases

in the delayed parenchymal phase following administration of

Sonazoid using a destructive mode technique (agent detection

imaging). Clin Radiol 63:1112–1120

6. Tamasi F et al (2005) ECHOVIST-200 enhanced hystero-

sonography: a new technique in the assessment of infertility. Eur

J Obstet Gynecol Reprod Biol 121:186–190

7. Rasor JS, Tickner EG (1984) Microbubble precursors and

methods for their production and use. US Patent 4,442,843

8. Uggowitzer MM et al (1999) Cerebral arteriovenous malforma-

tions: diagnostic value of echo-enhanced transcranial Doppler

sonography compared with angiography. AJNR Am J Neurora-

diol 20:101–106

9. Shiina Y et al (2009) Suitable solutions for reconstituting the

ultrasound contrast agent ‘‘Levovist’’ used in contrast echocar-

diogram: in vitro and in vivo evaluation of the influence of

osmotic pressure. Int J Cardiol 136:335–340

10. Nanda NC et al (2002) Multicenter evaluation of SonoVue for

improved endocardial border delineation. Echocardiography

19:27–36

11. Regouw BJM et al (1971) Specific determination of free fatty

acid in plasma. Clin Chim Acta 31:187–195

12. Davies JT, Rideal EK (1963) Interfacial Phenomena, 2nd ed.

Academic Press, NY

13. Fritzsch T et al (1990) Sonographic contrast medium with fatty

acid-containing microparticles and its preparation. Schering A.-G.,

Germany, p 7 (Application: DE)

14. Fritzsch T, Schartl M, Siegert J (1988) Preclinical and clinical

results with an ultrasonic contrast agent. Invest Radiol 23(Suppl

1):S302–S305

15. Makyla K, Paluch M (2009) The linoleic acid influence on

molecular interactions in the model of biological membrane.

Colloids Surf B 71:59–66

16. Supardan MD et al (2006) Use of ultrasonic technique for mea-

suring interfacial area in a two-dimensional bubble column.

J Chem Eng Jpn 39:687–692

17. Gilman JB et al (2004) Selectivity and stability of organic films at

the air-aqueous interface. J Colloid Interface Sci 280:234–243

18. Sherwin CP, Smith DE, Fulcher RG (1998) Effect of fatty acid

type on dispersed phase particle size distributions in emulsion

edible films. J Agric Food Chem 46:4534–4538

19. Goldenberg LC, Hutcheon I, Wardlaw N (1989) Experiments on

transport of hydrophobic particles and gas bubbles in porous

media. Transp Porous Media 4:129–145

20. Gonzenbach Urs T et al (2006) Stabilization of foams with

inorganic colloidal particles. Langmuir 22:10983–10988

21. Ross VE (1997) Particle-bubble attachment in flotation froths.

Miner Eng 10:695–706

22. Stechemesser H, Nguyen AV, Partzscht H (1998) Induction time

in particle/gas bubble interaction. Theory and experiment. Frei-

berg Forschungsh A841 (Partikeltechnologie):177–190

23. Kundu S (2008) Langmuir-Blodgett film from a bimolecular layer

at air-water interface. Colloids Surf A 317:618–624

24. Kundu S, Langevin D (2008) Fatty acid monolayer dissociation

and collapse. Effect of pH and cations. Colloids Surf 25:81–85

25. Sakai K, Takagi K (1994) Observation of coexistence of gas and

condensed phases in Langmuir films by scanning ripplon light

scattering technique. Langmuir 10:802–806

26. Seok S et al (2009) Imaging of collapsed fatty acid films at air-

water interfaces. Langmuir 25:9262–9269

27. Lee YH et al (2009) Effect of ultrasonic treatment on swine

wastewater solubilization. Water Sci Technol 59:603–608

Author Biographies

Lars Hoff received his master’s degree in physics from the

Norwegian Institute of Technology in 1989 and his Ph.D. from The

Norwegian University of Science and Technology in 2000. From

1990 to 1997 he worked as a research scientist at Nycomed

Amersham ASA in Oslo. Since 2003 he has been employed at

Vestfold University College, where he now is a full professor in micro

and nano systems technology.

Per A. Foss received his master’s and Ph.D. in bio-organic chemistry

in the period 1975–1985 at The Norwegian University of Science and

Technology. He was a postdoc at MIT in 1986–1987, and worked at

Nycomed Amersham/GE Healthcare in Oslo where he had various

positions. After a short period as CEO at Birkeland Innovation AS he

is now CEO at Hunt Biosciences AS in Norway.

Knut Dyrstad received his master’s degree in analytical chemistry at

The University of Oslo in 1992. The same year he was employed at

Nycomed Amersham ASA in Oslo. In 1998 he received his Ph.D. in

chemometrics, and is now a senior scientist at GE Healthcare in Oslo.

Jo Klaveness received a master’s degree in both pharmacy and

chemistry at The University of Oslo, and finally a Ph.D. in chemistry

in 1984. The same year, he was employed as a research chemist at

Nycomed Amersham ASA. He then founded Drug Discovery

Laboratory AS where he is now CEO. After various positions at

Nycomed, he is employed in his present position, full professor in

medicinal chemistry at The University of Oslo.

Pal Rongved received his master’s degree in chemistry in 1981. The

same year he was employed as a research chemist in Nycomed

Amersham ASA. In 2000 he received his Ph.D. in medicinal

chemistry. After various positions in Nycomed he was employed as

an innovation adviser for Birkeland Innovation AS, and now is an

associate professor in medicinal chemistry at the same institution.

J Surfact Deterg (2011) 14:585–593 593

123